Method for promoting diversification of antibody variable region

ABSTRACT

A method for promoting diversification of variable regions of an antibody, particularly a method for promoting diversification of the amino acid sequences of variable regions of an antibody generated by an avian B cell population, the method including suppressing the PI3Kα activity of each avian B cell comprised in the avian B cell population expressing the antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/484,061, filed Aug. 6, 2019, which is the U.S. National PhaseApplication of PCT International Application Number PCT/JP2018/004697,filed on Feb. 9, 2018, designating the United States of America andpublished in the Japanese language, which is an InternationalApplication of and claims the benefit of priority to Japanese PatentApplication No. 2017-023001, filed on Feb. 10, 2017. The disclosures ofthe above-referenced applications are hereby expressly incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via Patent Center ishereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e).The name of the ASCII xml file for the Sequence Listing isSEQLISTING_WNGR001.002APC.xml, the date of creation of the ASCII textfile is Jan. 2, 2023, and the size of the ASCII xml file is 21,653bytes.

TECHNICAL FIELD

The present invention relates to a method for promoting diversificationof the variable regions of an antibody.

BACKGROUND ART

An antibody binds to a specific antigen in a living body and provokesvarious biological defense reactions. Utilizing such properties of anantibody, various antibody drugs have been developed. In order todevelop such antibody drugs, a technique of producing many types ofantibodies, which bind with desired affinity to various antigens, isrequired.

In general, antibody production methods are classified into methods ofutilizing animal immunization and methods of not utilizing animalimmunization. As a method of utilizing animal immunization, a hybridomamethod comprising immunizing an animal with an antigen and then fusingthe obtained B cells with myelomas has been applied, for example.However, this method has been problematic in that time and effort arerequired to obtain an antibody because animals are used, and in that anantibody may not be obtained due to immune tolerance. As a method of notutilizing animal immunization, a phage display method has been applied,for example. This is a method comprising presenting a single chainantibody consisting of an antibody variable region (single chainvariable fragment; scFv) to phage particles to obtain a clone binding toa target antigen. However, this method has been problematic in that thequality of a library depends on the diversity of scFv, and in thatchanges are generated in specificity or affinity in the process ofconverting scFv to a full-length antibody.

In addition to the above-described techniques of producing antibodies, atechnique of producing antibodies by utilizing a chicken B cell-derivedDT40 cell line, ADLib (registered trademark) System, has been developed,and it has become possible to utilize a library capable of generatinghuman antibodies according to gene transfer (Patent Literatures 1, 2 and3, and Non Patent Literature 1). Since a clone having an antibodyspecifically binding to an antigen can be selected from a libraryaccording to this system, immune tolerance can be avoided, and afull-length antibody can be promptly obtained. What is more, since themechanism of the diversification of antibody variable regions, which isimportant for recognition of antigens, is gene conversion (GC),differing from V(D)J recombination in mice or humans, this system isadvantageous in that changes in antibody gene sequences can be expectedaccording to a mechanism different from antibodies produced in theliving bodies of mice or humans.

Several methods for diversifying the sequences of variable region genesof an antibody have been reported so far. Examples of such a methodinclude: animal immunization of using GANP mice (registered trademark),in which many somatic mutations are induced in the antibody variableregions of germinal center B cells, in comparison to normal wild-typemice (Patent Literature 4); and methods of utilizing avian B cellscomprising an inactivated XRCC3 gene (Patent Literature 5), or avian Bcells in which the expression of an AID gene has been controlled (PatentLiterature 6), or a DT40-SWΔC cell line, into which mutant AID withenhanced AID activity associated with mutation introduction has beenintroduced (Non Patent Literature 2).

Among others, the AID (activation-induced deaminase) protein has animportant function in causing somatic hypermutation (SHM) associatedwith the diversity of antibodies or maturation of affinity, class switchrecombination (CSR) for changing the class of the constant region of anantibody, and gene conversion (GC), and thus, a large number of studieshave been conducted regarding AID. When AID is associated with differentphenomena such as somatic hypermutation or class switch recombination,what mechanism regulates the functions of the AID is an extremelyinteresting issue. As a result of an experiment using AID mutants, ithas been reported that the C-terminus of AID is important for classswitch recombination but is not necessary for somatic hypermutation orgene conversion (GC) (Non Patent Literature 3). Moreover, it has beensuggested that different factors be recruited by AID, and then, therecruited factors induce different phenomena such as class switchrecombination or somatic hypermutation (Non Patent Literature 4).

PI3 Kinase (phosphoinositide 3-kinase: PI3K) has been known as a factorfunctioning upstream of AID. PI3 Kinase phosphorylates the position 3 ofthe inositol ring of phosphatidylinositol, and plays an important rolein various cellular functions such as cell survival, cell growth, cellmotility, and the transport of intracellular organelle. Such PI3 kinaseis classified into three classes (i.e., classes I, II, and III) in termsof structure. Class I is further divided into classes IA and IB, and ofthese, regarding the class IA, three isotypes p110α, p110β and p110δhave been known as catalytic subunits.

With regard to the relationship of PI3K with AID, as a result of studiesusing a PI3K inhibitor in mouse B cells, it has been demonstrated thatwhen p110δ signaling is suppressed, the expression of AID is increasedand class switch recombination is promoted (Non Patent Literature 5 andNon Patent Literature 6).

As described above, various factors are complicatedly involved in thediversification of the variable region genes of an antibody, and thecontrol mechanism thereof still remains unknown in many respects.

CITATION LIST Patent Literature Patent Literature 1: Japanese Patent No.4214234 Patent Literature 2: WO2008/047480 Patent Literature 3:WO2015/167011 Patent Literature 4: Japanese Patent No. 4478577 PatentLiterature 5: JP Patent Publication (Kokai) No. 2009-060850 A PatentLiterature 6: JP Patent Publication (Kokai) No. 2006-109711 A Non PatentLiterature 1: Seo et al., Nature Biotechnol. 23, 731-735 2005 Non PatentLiterature 2: Yuichi KANEHIRO et al., Summary of Annual Meeting of theSociety for Biotechnology, Japan, p. 109, 2008

Non Patent Literature 3: Barreto et al., Mol. Cell 12, 501-508, 2003

Non Patent Literature 4: Heltemes-Harris et al., Mol Immuno. 45,1799-1806, 2008 Non Patent Literature 5: Omori et al., Immunity 25,545-557, 2006 Non Patent Literature 6: Zhang et al., J Immunol. 191,1692-1703, 2013 Non Patent Literature 7: Backer, Curr Top MicrobiolImmunol. 346, 87-114, 2010 SUMMARY OF INVENTION Technical Problem

Taking into consideration the above-described circumstances, it is anobject of the present invention to provide a method for promotingdiversification of the amino acid sequences of antibody variable regionsand/or the gene sequences of antibody variable regions, which aregenerated by an avian B cell population.

Solution to Problem

The present inventors have screened for a drug for diversifying theamino acid sequences (complementarity-determining regions (CDR 1-3)) anda framework region (FR) of antibody variable regions (heavy chain (Hchain) and light chain (L chain)). As a result, the present inventorshave found that an inhibitor specific to the p110α isotype of a PI3kinase catalytic subunit (hereinafter referred to as “PI3Kα”) iseffective for the diversification of the amino acid sequences ofantibody variable regions. As mentioned above, it had been reportedthat, in mouse B cells, an inhibitor specific to the p110δ isotype(hereinafter referred to as “PI3Kδ”) promotes class switchrecombination. However, the present inventors have confirmed that theinhibitor to PI3Kδ hardly had influence on the diversification of theamino acid sequences of antibody variable regions of avian B cells.

In view of the above, it has been suggested that signals from PIK3α andPIK3δ are likely to be associated with the diversification of theantibody variable regions and class switch recombination in antibodyconstant regions, in each different pathways, or in collaboration withdifferent factors, or that pathways for inducing changes to the antibodysequences of B cells are different between mice and birds.

The present invention has been completed based on the aforementionedfindings.

Specifically, the present invention includes the following (1) to (9).

(1) A method for promoting diversification of the amino acid sequencesof variable regions of an antibody generated by an avian B cellpopulation, wherein the method comprises suppressing the PI3Kα activityof each avian B cell comprised in the avian B cell population expressingthe antibody.(2) The method according to the above (1), which is characterized inthat the avian B cell is a chicken B cell.(³) The method according to the above (2), which is characterized inthat the chicken B cell is a DT40 cell.(4) The method according to any one of the above (1) to (3), which ischaracterized in that the antibody variable region is an antibody heavychain variable region.(5) The method according to any one of the above (1) to (3), which ischaracterized in that the antibody variable region is an antibody lightchain variable region.(6) The method according to any one of the above (1) to (5), which ischaracterized in that the suppression of the PI3Kα activity is inducedby allowing a PI3Kα-specific inhibitor to come into contact with theavian B cell.(7) The method according to the above (6), which is characterized inthat the PI3Kα-specific inhibitor is any one of PI3Kα Inhibitor 2 andA66.(8) The method according to any one of the above (1) to (7), which ischaracterized in that the antibody expressed by the avian B cell is IgMor IgG.(9) The method according to any one of the above (1) to (8), which ischaracterized in that the antibody expressed by the avian B cell is anavian antibody, a chimeric antibody, a humanized antibody, or a humanantibody.

Advantageous Effects of Invention

According to the method of the present invention, it becomes possible topromote diversification of the amino acid sequences of variable regionsof an antibody generated by an avian B cell population, and thus, anantibody reacting against a desired antigen can be easily prepared.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a summary of a method for evaluating the diversification ofantibody variable regions according to flow cytometry.

FIG. 2 shows an example of analyzing antibody variable regions using thePI3K inhibitor Wortmannin. “CS(−)” indicates an example of performingthe analysis under drug non-additive conditions.

FIG. 3 shows comparison (1) in terms of the effects of PI3K inhibitorshaving different specificity on the diversification of antibody variableregions. As such inhibitors, Pan-inhibitors (exhibiting inhibitoryeffects on all of PI3Kα, β, δ and γ), PI3Kα-specific inhibitors, aPI3Kδ-specific inhibitor, and PI3Kγ-specific inhibitors were used. Thelongitudinal axis indicates the percentage of cIgM+_Sema3A− cells.

FIG. 4 shows comparison (2) in terms of the effects of PI3K inhibitorshaving different specificity on the diversification of antibody variableregions. As such inhibitors, PI3Kα-specific inhibitors, a PI3Kδ-specificinhibitor, and PI3Kβ-specific inhibitors were used. The longitudinalaxis indicates the percentage of cIgM+_Sema3A− cells.

FIG. 5 shows a comparison in terms of the effects of PI3Kα inhibitors onthe diversification of antibody variable regions. As such inhibitors,PI3Kα-specific inhibitors and drugs having PI3Kα inhibitory activitywere used. The longitudinal axis indicates the percentage ofcIgM+_Sema3A− cells.

FIG. 6 shows the verification results of light chain GC according toReversion assay. “CS(−)” indicates a drug non-additive control. CAL-101:PI3Kδ-specific inhibitor, CZC 24832: PI3Kγ-specific inhibitor, AS604850: PI3Kγ-specific inhibitor, A66: PI3Kα-specific inhibitor, andPI3Kai2 (PI3Kα Inhibitor 2): PI3Kα-specific inhibitor.

FIG. 7 shows the diversity analysis results of the amino acid sequencesof antibody heavy chain variable regions according to NGS analysis.

FIG. 8 shows the diversity analysis results of the amino acid sequencesof antibody light chain variable regions according to NGS analysis.

FIG. 9 shows an example of analyzing cell groups, the antigen-bindingability of which has been improved as a result of Affinity Maturation.The symbol “−”: drug non-additive conditions, A66: conditions comprisingPI3Kα-specific inhibitor A66, and PI3Kai2: conditions comprisingPI3Kα-specific inhibitor PI3Kα Inhibitor 2.

FIG. 10 shows the percentage of cells, the antigen-binding ability ofwhich has been improved as a result of Affinity Maturation. The symbol“−”: drug non-additive conditions, A66: conditions comprisingPI3Kα-specific inhibitor A66, and PI3Kai2: conditions comprisingPI3Kα-specific inhibitor PI3Kα Inhibitor 2.

DESCRIPTION OF EMBODIMENTS

An embodiment of the present invention relates to a method ofsuppressing the PI3Kα activity of individual avian B cells comprised inan avian B cell population that expresses antibodies, so as to promotediversification of the amino acid sequences of variable regions ofantibodies (antibody group) generated by the avian B cell population.

The term “avian B cells” in the phrase “avian B cell population (aplurality of avian B cells)” used in the present embodiment means avianB cells that generate antibodies. The avian B cells may be, for example,DT 40 cells as a chicken B cell-derived cell line, but are notparticularly limited thereto. Moreover, examples of the avian B cellsalso include avian B cells, on which a treatment of introducing avariety of mutations has been performed, such as avian B cells, in whichan XRCC3 gene has been inactivated (JP Patent Publication (Kokai) No.2009-060850 A), avian B cells, in which the expression of an AID genehas been controlled (JP Patent Publication (Kokai) No. 2006-109711 A),avian B cells, in which an antibody sequence has been diversified usinga HDAC (Histone Deacetylase) inhibitor comprising TSA (Trichostatin A),and avian B cells, in which a foreign gene sequence or a portion thereofhas been introduced onto the chromosome thereof (e.g., avian B cells,into which any given antibody gene sequence, etc. has been introduced).

The culture, etc. of the “avian B cells” used in the present embodimentcan be easily carried out according to a method well known to a personskilled in the art, and thus, the culture conditions, etc. are notparticularly limited. For example, in a case where the avian B cells areDT40 cells, IMDM medium (Invitrogen), etc. may be used, and the cellsmay be cultured in the presence of about 5% CO₂ at approximately 39.5°C.

In addition, the above-described “avian B cell population” may also havebeen cultured, in advance, in the presence of a calcineurin inhibitor(for example, FK506, etc.) and avian serum, before it is allowed to comeinto contact with an antigen. Otherwise, after completion of AffinityMaturation (which means, in the present description, a method forproducing a clone or a clone group, whose binding ability to an antigenis not only enhanced, but which has a change to improve the propertiesof an antibody, such as the improvement of physical properties), whenthe avian B cell population is allowed to come into contact with anantigen, it may be cultured in the presence of a calcineurin inhibitor(for example, FK506, etc.) and avian serum.

In the present embodiment, the “avian B cell population that generatesantibodies” includes avian B cells generating membrane antibodies andsecreted antibodies, and avian B cells generating membrane antibodies orsecreted antibodies.

In the present embodiment, the “antibody which the avian B cellsgenerate” may be an avian antibody, a chimeric antibody, a humanizedantibody, or a human antibody. With regard to the shape of an antibody,a full-length antibody, an antibody fragment (for example, F(ab′)2,Fab′, Fab, Fv, scFv, Fc, etc.), and a protein comprising the amino acidsequences of the variable regions of the heavy chain and/or light chainof an antibody, can be used, but are not limited thereto. Moreover, theantibody may also be a multispecific antibody (for example, a bispecificantibody), an antibody fragment thereof, or the like.

In the present description, the chimeric antibody is an antibody formedby linking regions having different origins to each other, and examplesof the chimeric antibody include an antibody formed by linking avariable region to a constant region, the origins of which are differentfrom each other, and an antibody formed by linking a Fab region to a Fcregion, the origins of which are different from each other, but are notlimited thereto. For instance, a bird-mouse chimeric antibody is anantibody formed by linking the amino acid sequence of an aviangene-derived antibody to the amino acid sequence of a mouse gene-derivedantibody. Other examples of the chimeric antibody include: a bird-humanchimeric antibody formed by linking the amino acid sequence of an aviangene-derived antibody to the amino acid sequence of a human gene-derivedantibody; a bird-rabbit chimeric antibody formed by linking the aminoacid sequence of an avian gene-derived antibody to the amino acidsequence of a rabbit gene-derived antibody; and a bird-goat chimericantibody formed by linking the amino acid sequence of an aviangene-derived antibody to the amino acid sequence of a goat gene-derivedantibody.

The humanized antibody is an antibody, in which among the amino acidsequences of the heavy or light chains of a generated antibody, somesequences are avian gene-derived sequences, and other sequences arehuman gene-derived sequences. On the other hand, the human antibody isan antibody, in which all of the amino acid sequences of the heavy orlight chains of a generated antibody are human gene-derived sequences.

Furthermore, in the present embodiment, the “antibody which the avian Bcells generate” includes antibodies comprising the entire or a part ofthe amino acid sequences of antibodies derived from animal species otherthan birds. Specific examples include, but are not limited to,antibodies comprising the entire or a part of the amino acid sequencesof antibodies obtained from a mouse, a rat, a rabbit, a bovine, a goat,and the like.

In the present embodiment, the isotypes of the “antibody which the avianB cells generate” are not particularly limited. Examples of such anisotype include IgM, IgG, IgA, and IgY.

In the present embodiment, the “variable region of an antibody” may beeither the variable region of an antibody heavy chain (H chain) or thevariable region of an antibody light chain (L chain). The variableregion of an antibody is a region consisting ofcomplementarity-determining regions (CDR 1-3) and a framework region(FR).

In the present embodiment, the “diversification” of the amino acidsequences of variable regions of an antibody means that, when the aminoacid sequences of the variable regions of antibodies, which individualavian B cells constituting an avian B cell population generate, arecompared with each other, the percentage of sequences identical to eachother is decreased, and sequences different from each other (inparticular, sequences having a low degree of similarity to each other)is increased. Examples of the indicator of the “diversification” of theamino acid sequences of variable regions that can be used herein mayinclude, but are not particularly limited to, the antigen specificity ofan antibody generated by the avian B cell population as a target, achange in antigen-binding ability, physical properties and the like, andthe number of the types of the amino acid sequences of antibody variableregions and identity or homology among the sequences. Further, since theamino acid sequences of antibody variable regions are univocallydetermined by the DNA sequences thereof, the diversity thereof can alsobe determined using, as an indicator, the number of the types of the DNAsequences of the variable regions of antibodies generated by the avian Bcell population as a target, identity or homology among the sequences,etc.

When the amino acid sequences of antibody variable regions of individualcells constituting the avian B cell population are diversified, changesare generated in the antigen specificity, affinity, physical propertiesand the like of antibodies generated by individual avian B cells, sothat the diversification of antibodies obtained from the avian B cellpopulation can be achieved.

The “PI3Kα” of the present embodiment means PI3 kinase catalytic subunitα isotype (p110α) (Non Patent Literature 7). Herein, examples of themethod of suppressing the activity of PI3Kα may include, but are notlimited to, a method of using a PI3Kα-specific activity inhibitor and amethod of reducing or eliminating the function of a PI3KCA gene encodingPI3Kα.

The method of using a PI3Kα-specific activity inhibitor can be carriedout, for example, by allowing a PI3Kα-specific inhibitor such as PI3KαInhibitor 2, A66, PF-4989216, INK1117, GSK1059615, GDC-0941, BYL719,PI-103, PIK-90, PIK-75 or HS-173 to come into contact with avian Bcells, and thus treating the cells. The method of allowing thePI3Kα-specific inhibitor to come into contact with the avian B cellscould be easily selected from known techniques by a person skilled inthe art. For example, a method of culturing avian B cells in a state inwhich a PI3Kα-specific inhibitor is present in a medium may be applied.The concentration of the PI3Kα-specific inhibitor in the medium isdifferent depending on the type of the inhibitor used. A person skilledin the art could easily determine the effective concentration of thePI3Kα-specific inhibitor within a range that does not damage the avian Bcells, by performing preliminary experiments and the like. When the usedPI3Kα-specific inhibitor is, for example, PI3Kα Inhibitor 2, theinhibitor may be added into a medium to a concentration of approximately500 nM to 5 nM. Likewise, in the case of using A66, the concentrationmay be approximately 10 μM to 100 nM; in the case of using PF-4989216,the concentration may be approximately 500 nM to 50 nM; in the case ofusing INK1117, the concentration may be approximately 2.5 μM to 25 nM;in the case of using GSK1059615, the concentration may be approximately500 nM to 5 nM; in the case of using GDC-0941, the concentration may beapproximately 500 nM to 5 nM; in the case of using BYL719, theconcentration may be approximately 500 nM to 5 nM; in the case of usingPI-103, the concentration may be approximately 50 nM to 5 nM; in thecase of using PIK-90, the concentration may be approximately 100 nM to10 nM; in the case of using PIK-75, the concentration may beapproximately 5 nM to 0.5 nM; and in the case of using HS-173, theconcentration may be approximately 50 nM to 5 nM.

Moreover, the PI3Kα-specific inhibitor is not particularly limited, aslong as it specifically inhibits the activity of PI3Kα. ThePI3Kα-specific inhibitor may be any one of a low-molecular-weightcompound, a protein, and a peptide, and also, a commercially availableproduct can be purchased and used.

The time necessary for allowing the PI3Kα-specific inhibitor to comeinto contact with the avian B cells and treating the cells with theinhibitor is different, depending on the type of the inhibitor used andthe concentration thereof. For example, when the above-describedPI3Kα-specific inhibitor is used, the necessary time may beapproximately 24 to 72 hours.

Examples of the method of reducing or eliminating the function of aPI3KCA gene may include a method of reducing or eliminating the activityof PI3Kα by performing genetic manipulation (e.g., introduction of pointmutation, or mutation such as deletion, insertion or addition; andgenome editing by knock-in, knock-out or CRISPER/Cas system) on a PI3KCAgene; a method of reducing or eliminating the expression of PI3Kα; and amethod of reducing or eliminating the expression of PI3Kα, using shRNA,etc. (RNAi method).

The disclosures of all publications cited in the present description areincorporated herein by reference in their entirety. In addition,throughout the present description, when singular terms such as “a,”“an,” and “the” are used, these terms include not only single items butalso multiple items, unless otherwise clearly specified.

Hereinafter, the present invention will be further described in thefollowing examples. However, these examples are only illustrativeexamples of the embodiments of the present invention, and thus, are notintended to limit the scope of the present invention.

EXAMPLES 1.Materials and Methods 1-1. Test Subjects, Samples, and UsedMaterials 1-1-1. Cell Lines

In the present Examples, the chicken-derived B cell lines, DT40 cells,namely, a BMAA4-2 cell line (WO2014/123186), a CL18_M− cell line (theCL18 cell line used in the present Examples; Buerstedde et al., EMBO J.9, 921-927, 1990), an hVEGF-A#33 cell line, and an hVEGF-A#44 cell linewere used. The BMAA4-2 cell line includes cells generating ananti-Sema3A antibody (chicken IgM: cIgM), the hVEGF-A#33 cell line andthe hVEGF-A#44 cell line include cells generating an anti-hVEGF antibody(human IgG: hIgG), and the CL18_M− cell line has a frame shift in thecIgM antibody gene region thereof, and thus, in general, does notgenerate the cIgM antibody.

The BMAA4-2 cell line was produced according to the method described inWO2014/123186. The hVEGF-A#33 cell line and the hVEGF-A#44 cell linewere selected from a human ADLib library (a library modified inaccordance with the method described in WO2015/167011), using humanVEGF-A (hereinafter referred to as “hVEGF-A”) as an antigen, and wereeach isolated as clones specifically binding to hVEGF-A. The hVEGF-A#33cell line has a light chain λ chain, whereas the hVEGF-A#44 cell linehas a light chain κ chain. These are cell lines generating antibodiesderived from different antibody gene sequences.

1-1-2. Medium, Serums, Antibiotic, Etc.

For the culture of DT40 cells, Iscove' s Modified Dulbecco' s Medium(IMDM, Gibco, 12440079) was used as a medium, and as serums, FetalBovine Serum (FBS, Biosera, FB1280/500, lot. 11824) and Chicken Serum(CS, Gibco, 16110082, lot. 1383279) were used. Also, as an antibiotic,Penicillin-Streptomycin Mixed Solution (Stabilized) (P/S, NacalaiTesque, 09367-34) was used. In addition, after completion of AffinityMaturation, FK-506 (Cayman, 10007965) was added and used.

1-1-3. Antigens and Antibodies

As antigens used in analyzing the diversification of antibodies, acynomolgus monkey Sema3A protein fused with a His tag and an AP tag(His-AP-cySema3A, NCBI Reference Sequence: XP_005550410.1) and anhVEGF-A protein fused with a FLAG tag (27-191 amino acid sequence,UniProt# P15692-4) were prepared. Antibodies used in the analysis wereMouse Anti-Chicken IgM-PE (SouthernBiotech, 8310-09), Goat anti-ChickenIgM Antibody FITC Conjugated (Bethyl, A30-102F), and Goat Anti-HumanIgG-PE (SouthernBiotech, 2040-09).

1-1-4. Primers Used in Next-Generation Sequencing (Ngs) Analysis

Analysis of DNA sequences of antibody heavy chain variable regionsBHcF1; 5′-CTATGCGCCTTGCCAGCCCGCTCAGCGCTCTCTGCCCTTCC-3′ (SEQ ID NO: 1)A001HcR; 5′- CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTCGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 2) A002HcR; 5′-CGTATCGCCTCCCTCGCGCCATCAGACGCTCGACACGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 3) A003HcR;5′-CGTATCGCCTCCCTCGCGCCATCAGAGACGCACTCGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 4) A004HcR; 5′-CGTATCGCCTCCCTCGCGCCATCAGAGCACTGTAGCGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 5) A005HcR; 5′-CGTATCGCCTCCCTCGCGCCATCAGATCAGACACGCGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 6) A006HcR; 5′-CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGCGATGACTTCGGTCCCGTG- 3′(SEQ ID NO: 7) A007HcR; 5′-CGTATCGCCTCCCTCGCGCCATCAGCGTGTCTCTACGATGACTTCGGTCCCGTG-3′ (SEQ ID NO: 8)Analysis of DNA sequences of antibody light chain variable regionsA001NGSLCF; 5′- CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTCAGGTTCCCTGGTGCAGGC-3′ (SEQ ID NO: 9) A002NGSLCF; 5′-CGTATCGCCTCCCTCGCGCCATCAGACGCTCGACACAGGTTCCCTGGTGCAGGC- 3′(SEQ ID NO: 10) A003NGSLCF; 5′-CGTATCGCCTCCCTCGCGCCATCAGAGACGCACTCAGGTTCCCTGGTGCAGGC-3′ (SEQ ID NO: 11)A005NGSLCF; 5′- CGTATCGCCTCCCTCGCGCCATCAGATCAGACACGCAGGTTCCCTGGTGCAGGC-3′ (SEQ ID NO: 12) A006NGSLCF; 5′-CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGCAGGTTCCCTGGTGCAGGC- 3′(SEQ ID NO: 13) A007NGSLCF; 5′-CGTATCGCCTCCCTCGCGCCATCAGCGTGTCTCTACAGGTTCCCTGGTGCAGGC- 3′(SEQ ID NO: 14) A012NGSLCF; 5′-CGTATCGCCTCCCTCGCGCCATCAGCGAGAGATACAGGTTCCCTGGTGCAGGC-3′ (SEQ ID NO: 15)cmNGSLcR-3B; 5′-CTATGCGCCTTGCCAGCCCGCTCAGATGTCACAATTTCACGATGG-3′(SEQ ID NO: 16)

1-1-5. Reagents, Etc.

In the present Examples, the reagents shown in Table 1 to Table 3 wereused.

TABLE 1 List of Screened Drugs Company name, Drug Name Function CatalogNo. T2AA mono-ubiquitinated Sigma-Aldrich, SML0794 PCNA inhibition RI-1RAD51 inhibition Abeam, ab144558 B02 RAD51 inhibition Merk/Millipore,553525 Piperlongumine ROS increase Sigma-Aldrich, SML0221 IrinotecanTopoisomeraseI Cayman chemical, 14180 inhibition EtoposideTopoisomeraseII Cayman chemical, 12092 inhibition Lipopoly- TLR4 ligandSigma-Aldrich, L3024 saccharides from Escherichia coli O111:B4 Poly(I:C)TLR3 ligand InvivoGen, tlrl-pic ODN D-SL01 TLR9 ligand InvivoGen,tlrk-dsl01 Chicken CD40LG CD40 ligand ImmunoChemistry RecombinantTechnologies, 6602 Protein Chicken BAFF B-cell activation factorImmunoChemistry Recombinant Technologies, 6554 Protein FluocinonideGlucocorticoid Santa Cruz, sc-255177 Wortmannin PI3K inhibition Caymanchemical, 10010591 Rapamycin FKBP-12 inhibition Cayman chemical, 13346Calcitriol Vitamin D3 analog Cayman chemical, 71820 MethotrexateDihydrofolate Cayman chemical, 13960 reductase (DHFR) inhibition10058-F4 c-Myc-Max Cayman chemical, 15929 dimerization inhibitionCPI-203 Bromodomain- Cayman chemical, 15479 containing protein 4 (BRD4)inhibition PF-04691502 mTOR/PI3K inhibition Sigma-Aldrich, PZ0235PF-05212384 mTOR/PI3K inhibition Sigma-Aldrich, PZ0281 SCR7 pyrazine DNAligase IV Cayman chemical, 18015 inhibition Cdk2 Inhibitor II Cdk2inhibition Cayman chemical, 15154 Cdk4 Inhibitor Cdk4 inhibition Caymanchemical, 17648 Cdk1/5 Inhibitor Cdk1/5 inhibition Cayman chemical,18740 Cdk1/2 Cdk1/2 inhibition Cayman chemical, 18859 Inhibitor IIICdk4/6 Cdk4/6 inhibition Cayman chemical, 17974 Inhibitor IV

TABLE 2 PI3K Inhibitor Company name, Drug Name Function Catalog No.Wortmannin PI3K inhibition Cayman chemical, 10010591 LY294002 PI3Kinhibition Cayman chemical, 70920 A66 PI3Kα inhibition Sigma-Aldrich,SML1213 PI3Kα PI3Kα inhibition Cayman chemical, 10010177 Inhibitor 2INK1117 PI3Kα inhibition Cayman chemical, 19514 (MLN1117) GSK1059615PI3Kα inhibition Cayman chemical, 11569 BYL719 PI3Kα inhibition Caymanchemical, 16986 PIK-75 PI3Kα inhibition Cayman chemical, 10009210 HS-173PI3Kα inhibition Cayman chemical, 19156 PI-103 PI3Kα/DNA-PK Caymanchemical, 10009209 inhibition PIK-90 PI3Kα/γ inhibition Cayman chemical,10010749 PF-4989216 PI3Kα/δ inhibition Cayman chemical, 19308 GDC-0941PI3Kα/δ inhibition Cayman chemical, 11600 AZD6482 PI3Kβ inhibitionCayman chemical, 15250 TGX-221 PI3Kβ inhibition Cayman chemical,10007349 CZC 24832 PI3Kγ inhibition Sigma-Aldrich, SML1214 AS 604850PI3Kγ inhibition Sigma-Aldrich, A0231 AS-252424 PI3Kγ inhibitionSigma-Aldrich, A8981 CAL-101 PI3Kδ inhibition Cayman chemical, 15279IC-87114 PI3Kδ inhibition Cayman chemical, 11589

TABLE 3 List of screened Drugs Used in Human Antibody Producing CellLines Company name, Drug Name Function Catalog No. T2AAmono-ubiquitinated Sigma-Aldrich, SML0794 PCNA inhibition WortmanninPI3K inhibition Cayman chemical, 10010591 A66 PI3Kα inhibitionSigma-Aldrich, SML1213 AS-252424 PI3Kγ inhibition Sigma-Aldrich, A8981IC-87114 PI3Kδ inhibition Cayman chemical, 11589 INK1117 PI3Kαinhibition Cayman chemical, 19514 PI-103 PI3Kα/DNA-PK Cayman chemical,10009209 inhibition RI-1 RAD51 inhibition Abeam, ab144558 IrinotecanTopoisomeraseI Cayman chemical, 14180 inhibition CZC 24832 PI3Kγinhibition Sigma-Aldrich, SML1214 PIK-93 PI4KIIIβ inhibitionSigma-Aldrich, SML0546 AZD6482 PI3Kβ inhibition Cayman chemical, 15250GSK1059615 PI3Kα inhibition Cayman chemical, 11569 PIK-90 PI3Kα/γinhibition Cayman chemical, 10010749 B02 RAD51 inhibitionMerk/Millipore, 553525 LY294002 PI3K inhibition Cayman chemical, 70920AS 604850 PI3Kγ inhibition Sigma-Aldrich, A0231 Calcitriol Vitamin D3analog Cayman chemical, 71820 TGX-221 PI3Kβ inhibition Cayman chemical,10007349 GDC-0941 PI3Kα/δ inhibition Cayman chemical, 11600 PIK-75 PI3Kαinhibition Cayman chemical, 10009210 Piperlongumine ROS increaseSigma-Aldrich, SML0221 CAL-101 PI3Kδ inhibition Cayman chemical, 15279PI3Kα PI3Kα inhibition Cayman chemical, 10010177 Inhibitor 2 PF-4989216PI3Kα/δ inhibition Cayman chemical, 19308 BYL719 PI3Kα inhibition Caymanchemical, 16986 HS-173 PI3Kα inhibition Cayman chemical, 19156

1-2. Experimental Method 1-2-1. Composition of Media and Buffer

The following media and buffer were used.(i) CS(−) medium

IMDM: 1,000 mL FBS: 90 mL P/S: 10 mL

(ii) CS(+) medium

IMDM: 1,000 mL FBS: 90 mL P/S: 10 mL CS: 10 mL

(iii) FACS buffer

BSA: 5 g 0.5 M EDTA (pH 8.0): 4 mL

Phosphate Buffered Saline Powder: 1 unitdH₂O: 1,000 mL

0.22 μm Filter System (Corning, 431098).

1-2-2. Labeling of Antigen with Alexa Fluor 647 (Af647)

Using Alexa Fluor 647 Microscale Protein Labeling kit (Molecular probes,A30009), His-AP-cySema3A was labeled with AF647. Since the concentrationof the protein used herein, His-AP-cySema3A, was less than 1 mg/mL(i.e., 0.626 mg/mL), the dye: molar ratios (MR) were set at 25, and theamount of the dye was calculated. Regarding other points, theinstruction manuals included with the kit were applied. Using NanoDrop2000 c (ThermoFisher Scientific), A280 and A650 were measured, and theconcentration of the protein in the fluorescently labeled sample wasthen determined. The thus produced AF647-labeled Sema3A was used in thesubsequent experiments.

1-2-3. Sequence Change Analysis Using Bmaa4-2 Cell Line According toFlow Cytometry (Fcm)

A frozen stock of the BMAA4-2 cell line (3.0×10⁶ cells) was awaken with10 mL of CS(−) medium, using a 25 cm² Flask (Corning, 430639), and thecells were then cultured in a CO₂ incubator (SANYO CO₂ IncubatorMCO-20AIC, 39.5° C., 5% CO₂). On the following day, 500 μL each of aCS(−) medium, to which each drug had been added, was added into eachwell of a 48-well dish (Nunc, 150687), and the cells were then seededthereon to a cell density of 6.0×10⁴ cells/mL (wherein the cellconcentration and the survival rate were measured using CASY cellcounter (Nepa Gene)). After individual samples had been cultured for 2days, the samples were subcultured under the same conditions to resultin a cell density of 1.0×10⁴ cells/mL. After completion of the culturefor 3 days, individual samples were subcultured under the sameconditions to result in a cell density of 6.0×10⁴ cells/mL. Aftercompletion of the culture for 2 days (Day 7 of the culture), individualsamples (in each amount of 2.0×10⁵ cells) were transferred into eachwell of a 96-well plate (Nunc, 249662), and thereafter, a supernatantwas removed by centrifugation at 300× g for 5 min. 200 μL each of FACSbuffer was added into each well, and the cells were washed. After that,the resulting cells were centrifuged (300× g, 3 min) again, and asupernatant was then removed. This washing step was carried outrepeatedly twice, and each sample was then suspended in 50 μL of FACSbuffer comprising AF647-labeled Sema3A (5 nM) and 200-fold diluted MouseAnti-Chicken IgM-PE. The light was shielded, and the obtained suspensionwas then reacted at 4° C. for 30 min. After completion of the staining,the reaction mixture was washed with a FACS buffer twice, and eachsample was then suspended in 100 μL of FACS buffer comprising 1,000-folddiluted 7-AAD (BD Pharmingen, 559925), followed by measurement usingFACS Canto II Flow Cytometer. Using FSC-SSC plot, dead cells, doubletcells, and 7-AAD-positive cells were removed. Thereafter, a plot, inwhich the vertical axis indicated APC (AF647 -labeled Sema3A) and thelongitudinal axis indicated PE (Mouse Anti-Chicken IgM-PE), wasdeveloped, and the percentage of PE+(cIgM+)_APC-(Sema3A−) cells was thencalculated.

1-2-4. Light Chain Gc (Gene Conversion) Analysis According to ReversionAssay

A frozen stock of the CL18_M− cell line (3.0×10⁶ cells) was awaken with10 mL of CS(−) medium, using a 25 cm² Flask, and the cells were thencultured in a CO₂ incubator (39.5° C., 5% CO₂). On the following day, 2mL each of a CS(−) medium, to which each drug had been added, was addedinto each well of a 24 deep well plate (ThermoFisher Scientific,95040480), and the cells were then seeded thereon to a cell density of6.0×10⁴ cells/mL (wherein the cell concentration and the survival ratewere measured using CASY cell counter). After individual samples hadbeen cultured for 2 days, the samples were subcultured under the sameconditions to result in a cell density of 1.0×10⁴ cells/mL. Aftercompletion of the culture for 3 days, individual samples weresubcultured under the same conditions to result in a cell density of6.0×10⁴ cells/mL, and then, were further cultured for 2 days, so thatthe samples were cultured for a total of 7 days under the sameconditions. At the time point of Day 7 of the culture, individualsamples (in each amount of 2.0×10⁵ cells) were transferred into eachwell of a 96-well plate (Nunc, 249662), and thereafter, a supernatantwas removed by centrifugation at 300× g for 5 min. 200 μL each of FACSbuffer was added into each well, and the cells were washed. After that,the resulting cells were centrifuged (300× g, 3 min) again, and asupernatant was then removed. This washing step was carried outrepeatedly twice, and each sample was then suspended in 50 μL of FACSbuffer comprising 1,000-fold diluted Goat anti-Chicken IgM Antibody FITCConjugated. The light was shielded, and the obtained suspension was thenreacted at 4° C. for 30 min. After completion of the staining, thereaction mixture was washed with a FACS buffer twice, and each samplewas then suspended in 100 μL of FACS buffer comprising 1,000-folddiluted 7-AAD, followed by measurement using FACS Canto II FlowCytometer. Using FSC-SSC plot, dead cells, doublet cells, and7-AAD-positive cells were removed. Thereafter, a plot, in which thevertical axis indicated FITC (Goat anti-Chicken IgM Antibody FITCConjugated) and the longitudinal axis indicated PE (none), wasdeveloped, and the percentage of PE−_FITC+(cIgM+) cells was thencalculated.

Meanwhile, after completion of the culture for 7 days, individualsamples were subcultured under the same conditions to result in a celldensity of 6.0×10⁴ cells/mL. After completion of the culture for 2 days,the samples were subcultured under the same conditions to result in acell density of 1.0×10⁴ cells/mL. After completion of the culture for 3days, individual samples were subcultured under the same conditions toresult in a cell density of 6.0×10⁴ cells/mL. The resulting cells werefurther cultured for 2 days, so that the samples were cultured for atotal of 14 days under the same conditions. At the time point of Day 14of the culture, individual samples were measured using FACS Canto IIFlow Cytometer, as with the measurement on Day 7.

1-2-5. Sequence Change Analysis According to Ngs (Heavy Chain)

A frozen stock of the BMAA4-2 cell line (3.0×10⁶ cells) was awaken with10 mL of CS(−) medium, using a 25 cm² Flask, and the cells were thencultured in a CO₂ incubator (39.5° C., 5% CO₂). On the following day, 2mL each of a CS(−) medium, to which each drug had been added, was addedinto each well of a 12-well dish (Nunc, 150628), and the cells were thenseeded thereon to a cell density of 6.0×10⁴ cells/mL (wherein the cellconcentration and the survival rate were measured using CASY cellcounter). After individual samples had been cultured for 2 days, thesamples were subcultured under the same conditions to result in a celldensity of 1.0×10⁴ cells/mL. After completion of the culture for 3 days,individual samples were subcultured in a 25 cm² Flask to result in acell density of 6.0×10⁴ cells/mL with respect to 10 mL of the mediumunder the same conditions. After completion of the culture for 2 days(Day 7 of the culture), individual samples (in each amount of 2.0×10⁶cells) were recovered in a 1.5-mL tube, and thereafter, a supernatantwas removed by centrifugation at 1,000× g for 5 min. The residue wasre-suspended in 1 mL of D-PBS(−) (Nacalai Tesque, 14249-24), and asupernatant was then removed by centrifugation at 1,000× g for 5 min.The residue was preserved at −80° C. Moreover, individual samples fromthe remaining cells were subcultured in a 12-well dish to result in acell density of 6.0×10⁴ cells/mL with respect to 2 mL of the mediumunder the same conditions. After completion of the culture for 2 days,individual samples were subcultured under the same conditions to resultin a cell density of 1.0×10⁴ cells/mL. After completion of the culturefor 3 days, individual samples were subcultured in a 25 cm² Flask toresult in a cell density of 6.0×10⁴ cells/mL with respect to 10 mL ofthe medium under the same conditions. After completion of the culturefor 2 days (Day 14 of the culture), individual samples (in each amountof 2.0×10⁶ cells) were recovered in a 1.5-mL tube, and thereafter, asupernatant was removed by centrifugation at 1,000× g for 5 min. Theresidue was re-suspended in 1 mL of D-PBS(−), and a supernatant was thenremoved by centrifugation at 1,000× g for 5 min. The residue waspreserved at −80° C.

(Light Chain)

A frozen stock of the BMAA4-2 cell line (3.0×10⁶ cells) was awaken with10 mL of CS(−) medium, using a 25 cm² Flask, and the cells were thencultured in a CO₂ incubator (39.5° C., 5% CO₂). On the following day, 1mL each of a CS(−) medium, to which each drug had been added, was addedinto each well of a 24-well dish, and the cells were then seeded thereonto a cell density of 3.0×10⁵ cells/mL (wherein the cell concentrationand the survival rate were measured using CASY cell counter). On thefollowing days, individual samples were subcultured under the sameconditions to result in a cell density of 1.0×10⁴ cells/mL. Aftercompletion of the culture for 3 days, individual samples weresubcultured in a 6-well dish to result in a cell density of 3.0×10⁵cells/mL with respect to 5 mL of the medium under the same conditions.On the following days, individual samples were subcultured in a 50-mLtube (TPP, 87050) to result in a cell density of 3.0×10⁵ cells/mL withrespect to 10 mL of the medium under the same conditions. On thefollowing days, individual samples were subcultured again in a 50-mLtube to result in a cell density of 3.0×10⁵ cells/mL with respect to 10mL of the medium under the same conditions. On the following day (Day 7of the culture), individual samples were subcultured in a 24-well dishto result in a cell density of 3.0×10⁵ cells/mL with respect to 1 mL ofthe medium under the same conditions. On the following day, individualsamples were subcultured under the same conditions to result in a celldensity of 1.0×10⁴ cells/mL. After completion of the culture for 3 days,individual samples were subcultured in a 50-mL tube to result in a celldensity of 6.0×10⁴ cells/mL with respect to 10 mL of the medium underthe same conditions. After completion of the culture for 2 days,individual samples were subcultured under the same conditions to resultin a cell density of 3.0×10⁵ cells/mL. On the following day (Day 14 ofthe culture), individual samples (in each amount of 2.0×10⁶ cells) wererecovered in a 1.5-mL tube, and thereafter, a supernatant was thenremoved by centrifugation at 1,000× g for 5 min. The residue wasre-suspended in 1 mL of D-PBS(−), and a supernatant was then removed bycentrifugation at 1,000× g for 5 min. The residue was preserved at −80°C.

Using Wizard Genomic DNA Purification Kit (Promega, A2361), genomic DNAwas extracted and purified from the cell pellets preserved at −80° C.This genomic DNA was used as a template. For the analysis of the heavychain, using the primers BHcF1 and A001HcR-A007HcR, and also usingPrimeSTAR GXL DNA Polymerase (Takara bio, R050B), after denaturation hadbeen carried out at 98° C. for 1 min, a cycle consisting of denaturationat 98° C. for 10 sec, annealing at 58° C. for 15 sec, and an elongationreaction at 68° C. for 1 min was repeated 35 times. Finally, anelongation reaction at 68° C. for 5 min was carried out. According tothese protocols, PCR was carried out. On the other hand, for theanalysis of the light chain, using the primers A001NGSLCF-A003NGSLCF,A005NGSLCF-A007NGSLCF, A012NGSLCF and cmNGSLcR-3B, and also usingPrimeSTAR GXL DNA Polymerase, after denaturation had been carried out at98° C. for 1 min, a cycle consisting of denaturation at 98° C. for 10sec, annealing at 60° C. for 15 sec, and an elongation reaction at 68°C. for 1 min was repeated 35 times. Finally, an elongation reaction at68° C. for 5 min was carried out. According to these protocols, PCR wascarried out. Using QIAquick PCR Purification Kit (Qiagen, 28106) orQIAquick Gel Extraction Kit (Qiagen, 28706), each PCR product waspurified, and thereafter, the concentration was measured using NanoDrop.Individual samples (400 ng each) were mixed with one another, andthereafter, the obtained mixture was then subjected to 1% agarose gelelectrophoresis, and was then purified using QlAquick Gel ExtractionKit. In these operations, a washing step using PE buffer and thesubsequent drying step were carried out twice, while changing thedirection of the tube. The concentration of the purified DNA Mix samplewas precisely measured using Quant-iT PicoGreen dsDNA Assay Kit(ThermoFisher scientific, P7589). Using GS Junior Titanium emPCR Kit(Lib-A) (Roche, Cat. No. 05 996 520 001) in accordance with theinstruction manual included therewith, emulsion PCR was carried out.Then, using GS Junior Titanium Sequencing Kit (Roche, Cat. No. 05 996554 001) and GS Junior Titanium PicoTiterPlate Kit (Roche, Cat. No. 996619 001) in accordance with the instruction manuals included therewith,sequencing was carried out with GS junior (Roche Life science). Aftercompletion of the sequencing, base calling was carried out in ampliconmode, to obtain raw data (read sequences).

The read sequences outputted from the GS Junior were subjected tofiltering, and when the concerned sequence did not satisfy the followingthree conditions, it was excluded:

(1) a read sequence comprising 250 bp or more of consecutive nucleotidesof QV15 or more,(2) a read sequence, in which all frame works can be recognized byregion estimation, and(3) a read sequence, in which a stop codon or a frame shift does notappear in the translation frames from CDR1 to CDR3.

With regard to the region estimation, using HMMER program (3.1b2,Johnson et al., BMC Bioinformatics. 11, 431. 2010), each regionestimation profile of FW1, FW2, FW3 and FW4 according to the Kabatdefinition was hit, so as to determine the regions. The read sequences,in which any FW was not recognized, were excluded, and then, in theremaining read sequences, CDR1, CDR2, and CDR3 were determined based onthe estimated FW regions. After completion of the filtering, 239 readsequences were randomly subjected to sampling without replacement, andNUS (Number of Unique Sequence) and SBL (Sum of Branch Length) were thencalculated, targeting the amino acid sequences of CDR1 to CDR3. NUSindicates the number of unique amino acid sequences in the 239sequences, whereas SBL indicates the sum of the lengths of branches oflineage trees obtained from a group of those amino acid sequences. Forthe production of such lineage trees, first, multiple alignment wascarried out using mafft program (v7.221, Katoh & Standley, Mol. Biol.Evol. 4, 772-780. 2013), and further, a p distance matrix based on themultiple alignment results was used as input data, and a lineage treewas produced according to a neighbor-joining method, using clearcutprogram (1.0.9, Savolainen et al., Syst. Biol. 49, 306-362. 2000). Thesampling of the 239 sequences was independently carried out 10 times,and the mean value of the NUS SBL values in each trial was determined tobe the final output value.

1-2-6. Sequence Change Analysis of Anti-Hvegf-A Antibody Generating CellLines According to Affinity Maturation and Flow Cytometry (Fcm)

Frozen stocks of the hVEGF-A#33 cell line and the hVEGF-A#44 cell line(3.0×10⁶ cells) were each awaken with a CS(−) medium, using a 25 cm²Flask (Corning, 430639), and were then cultured in a CO₂ incubator(SANYO CO₂ Incubator MCO-20AIC, 39.5° C., 5% CO₂). On the following day,the cells were seeded on a 6-well dish (Nunc, 140675) to a cell densityof 1.0×10⁴ cells/mL (wherein the cell concentration and the survivalrate were measured using CASY cell counter (Nepa Gene)). The cells werecultured in a CS(+) medium, in the presence or absence of a PI3Kαinhibitor (A66: 10 μM, PI3Kα Inhibitor 2: 500 nM). Moreover, after thecells had been cultured for 2 days, individual samples were subculturedunder the same conditions to result in a cell density of 1.0×10⁴cells/mL. Further, after completion of the culture for 3 days,individual samples were subcultured under the same conditions to resultin a cell density of 6.0×10⁴ cells/mL, and the culture was continued.After completion of the culture for 2 days (Day 7 of the culture),individual samples were centrifuged to remove a supernatant, and theresidue was then suspended in a CS(+) medium supplemented with 1 μMFK-506 (hereinafter referred to as a “CS/FK medium”). The suspension wascentrifuged again to remove a supernatant. This washing step was carriedout repeatedly twice, and individual samples were then suspended in aCS/FK medium supplemented with AF647-labeled hVEGF-A (10 nM) and2,000-fold diluted Goat Anti-Human IgG-PE. The light was shielded, andthe obtained suspension was then reacted at 4° C. for 30 min. Aftercompletion of the staining, the reaction mixture was washed with a CS/FKmedium twice, and each sample was then suspended in the CS/FK medium.Thereafter, using FACS Aria Fusion (BD), FCM and single cell sorting wascarried out. Dead cells were removed using FSC-SSC plot. A plot, inwhich the vertical axis indicated AF647 (AF647-labeled hVEGF-A) and thelongitudinal axis indicated PE (Goat Anti-Human IgG-PE), was developed,and the percentage of PE+(hIgG+)/AF647-(hVEGF-A−) cells was thencalculated. In order to make a comparison regarding the number of cellswith improved antigen-binding ability according to Affinity Maturationunder individual conditions, Affinity Maturation Gate was establishedaccording to the following criteria. First, the plot was converted to acontour plot in FCM analysis, and a trapezoid gate having an inclinationthat was in parallel with the contour of a population in which the mostcells were gathered (hereinafter referred to as a “main population”) wasestablished. Next, from the contour plot of the main population, a meanvalue of the fluorescence intensity as an indicator of binding abilityto an antigen (mean fluorescence intensity (MFI)) was calculated, andcells exhibiting MFI that was 5 or more times higher than the calculatedmean value were set to enter the gate. At that time, the vertex of thetrapezoid gate was set to be overlapped with the mean value MFI of themain population, and then, a cell population having low antigen-bindingability and a cell population having a low expression level of antibodywere set not to enter the gate. The percentage of clones with improvedantigen-binding ability was calculated by counting the number of cellsexisting in this Affinity Maturation Gate and then dividing the obtainedvalue by the total number of the analyzed cells.

1-2-7. Obtainment of Antibody Variable Region Gene Sequences of Cloneswith Improved Antigen-Binding Ability According to Affinity Maturation,and Sequence Analysis

From a cell population that corresponded to the top 0.2% of a cellpopulation having antigen-binding ability that was improved afterAffinity Maturation, 192 cells were single-cell-sorted, and were thencultured in a CS/FK medium on a 96-well plate for 8 days. From the thuscultured and growing clones, 24 clones were randomly picked up, and thegene sequence of each antibody variable region was then analyzed.According to a method of dividing heavy and light chain variableregions, which encoded the same nucleic acid sequence, into one group,the grouping of the clones was carried out. From the number of groups,the number of sequence types was calculated, and a change in the aminoacid sequences was confirmed. In addition, with regard to the number ofsequence changes, the original clones of each cell line were comparedwith clones obtained after Affinity Maturation, in terms of the nucleicacid sequence of an antibody variable region gene, and when one or moredifferent nucleotide sequences were found, it was counted as a sequencechange.

1-2-8. Measurement of Antibody Affinity of Clone with ImprovedAntigen-Binding Ability According to Affinity Maturation

With regard to representative clones of each group, which was obtainedby the grouping in the above 1-2-7, a culture supernatant was prepared,and the affinity with an antigen was then measured using an SPR method(Biacore T200, GE Healthcare). Using Human Antibody Capture Kit(BR100839, GE Healthcare), an anti-human IgG (Fc) antibody wasimmobilized on a CM5 sensor chip (BR100530, GE Healthcare), and anantibody contained in the culture supernatant was then captured. 25 nMhVEGF-A was reacted therewith for 240 seconds, and then, was dissociatedtherefrom for 500 seconds. As a regeneration solution, 3 M MgCl₂ wasreacted with the resultant for 30 seconds, so that one cycle wascompleted. As a buffer, HBS−EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% (v/v) surfactant P20 (pH 7.4) (BR100669, GE Healthcare)) was used,and the measurement was carried out at a flow rate of 30 μL/min.Employing Biacore T200 Evaluation Software, fitting was carried out oneach SPR sensorgram according to a Langmuir 1:1 binding model, so as tocalculate the association rate constant kon and the dissociation rateconstant koff. Then, according to the equation: KD=koff/kon, the KDvalue was determined.

1-3. Results 1-3-1. Screening for Compound Promoting Diversification ofAntibody Variable Region Amino Acid Sequences, Using Bmaa4-2 Cell Line

Using the BMAA4-2 cell line, a system for screening for adiversification-promoting compound was constructed (FIG. 1 ). TheBMAA4-2 cell line includes DT40 cell clones expressing anti-Sema3Aantibodies (cIgM). However, if the BMAA4-2 cell line were cultured underconditions in which gene conversion (GC) or somatic hypermutation areinduced, a cell population with reduced affinity for Sema3A as anantigen would appear. It is considered that the affinity of such a cellpopulation for the antigen has been reduced as a result of changes inthe amino acid sequences of variable regions. Thus, the percentage ofcIgM+_Sema3A− populations under individual culture conditions ismeasured and compared according to FCM, so that the effect of promotingchanges in the amino acid sequences (i.e., the effect of promoting thediversification of the amino acid sequences) can be evaluated.

First, from compounds that had been known to be associated with cellgrowth, differentiation and/or activation of B cells, gene conversion(GC), somatic hypermutation and the like, the compounds shown in Table 1were selected. The selected compounds were each added into a medium andwere then cultured. Thereafter, using, as an indicator, an increase inthe cIgM+_Sema3A− populations according to FCM at the time point of 1week after the culture, a compound promoting changes in the amino acidsequences of variable regions was screened. As a result, when Wortmanninas an inhibitor of PI3K was added, the percentage of the cIgM+_Sema3A−populations was increased in comparison to a negative control (CS(−)),and thus, Wortmannin was found to promote changes in the amino acidsequences of antibody variable regions (FIG. 2 ).

1-3-2. Comparison of Pi3K Inhibitors in Terms of Action on Changes inAmino Acid Sequences of Antibody Variable Regions, Using Bmaa4-2 CellLine

It was found that the PI3K inhibitors have an action to promote thediversification of the amino acid sequences of antibody variableregions. Thus, next, the inhibitors shown in Table 2 were selected fromamong various PI3K inhibitors exhibiting different isotype specificity,and these compounds were then compared with one another, in terms ofaction on sequence change. The concentration of each inhibitor wasdetermined, using IC₅₀ to acted molecules as a reference, according to apreliminary experiment, in which the presence or absence of isotypespecificity and cytotoxicity was used as an indicator. Using the BMAA4-2cell line, changes in the cIgM+_Sema3A− populations were compared amongindividual inhibitors according to FCM. As a result, it was found thatthe PI3Kα-specific inhibitors A66 and PI3Kα Inhibitor 2 had the highestsequence change-promoting action, and that the sequence change-promotingaction of a PI3Kβ-specific inhibitor, a PI3Kδ-specific inhibitor and aPI3Kγ-specific inhibitor was lower than that of A66 or PI3Kα Inhibitor2, or was almost at the same level as that of the negative control(CS(−)) (FIGS. 3 and 4 ).

Furthermore, the same experiment as described above was carried outusing PI3Kα-specific inhibitors and drugs having an inhibitory activityon PI3Kα. As a result, it was confirmed that the drugs inhibiting thePI3Kα activity have an action to promote sequence changes, in comparisonto the negative control (CS(−)) (FIG. 5 ). From these results, it becameclear that drugs inhibiting PI3Kα activity promote sequence changes.

1-3-3. Verification of Action Of Pi3Kα Inhibitor on Light Chain GeneConversion (Gc) According to Reversion Assay

The PI3Kα inhibitors such as A66 and PI3Kα Inhibitor 2 were found to bepromising as novel diversification-promoting compounds. Hence, theaction of these inhibitors was verified according to Reversion assaythat had been conventionally widely used to verify GC inducing activity.The Reversion assay is a system for verifying the frequency of GC,utilizing the phenomenon that a CL18 cell line having a frame shift inthe antibody light chain variable region thereof expresses IgM as aresult of GC (Reversion activity) (Buerstedde et al., EMBO J. 9,921-927. 1990). Specifically, when the percentage of cells expressingIgM is increased in the Reversion assay, it can be determined that GC isprovoked in the antibody light chain variable gene region, and that thediversification of the amino acid sequence of the region is therebypromoted. Since the CL18_M− cell line used in the present assay has theproperty of causing toxicity due to PI3K inhibitors, PI3Kα Inhibitor 2was used in a concentration of 200 nM in this experiment. The percentageof IgM expression-positive cells one and two weeks after the cultureperformed under individual conditions was calculated. As a result, itwas found that A66 and PI3Kα Inhibitor 2 used as PI3Kα inhibitorsexhibited higher Reversion activity than the PI3Kδ-specific inhibitor(CAL-101) and the PI3Kγ-specific inhibitors (CZC 24832 and AS 604850),and also, than a negative control (CS(−)) (FIG. 6 ).

1-3-4. Analysis of Antibody Variable Region Amino Acid Sequences OfPi3Kα Inhibitor-Treated Samples According to Ngs

Finally, using NGS, the antibody heavy chain variable region genes ofA66- and PI3Kα Inhibitor 2-treated samples were decoded, and alarge-scale analysis was then performed on the amino acid sequences ofthe obtained antibodies. Whether the diversity of antibodies wasimproved in the BMAA4-2 cell population was confirmed. Using either NUCrelative to the number of sequence types or SBL relative to the degreeof similarity among sequences as an indicator, the A66- and PI3KαInhibitor 2-treated samples exhibited higher values than the negativecontrol sample (FIG. 7 ).

Likewise, the antibody light chain variable region gene sequence of theA66-treated sample was also decoded, and a large-scale analysis was thenperformed on the amino acid sequence of the obtained antibody. As aresult, in both cases of using NUS and SBL, the A66-treated sampleexhibited higher values than the negative control sample (FIG. 8 ).

1-3-5. Screening for Compound Promoting Diversification of AntibodyVariable Region Amino Acid Sequence, Using Hvegf-A−#33 Cell Line

In order to select a diversification-promoting compound that waseffective for DT40 cells expressing a human antibody (hIgG), using aDT40 cell line expressing an anti-hVEGF-A antibody (hIgG) (hVEGF-A−#33cell line), and using, as an indicator, the percentage of a cellpopulation (hIgG+_hVEGF-A−) with reduced affinity for hVEGF-A serving asan antigen, compounds were screened in the same manner as that of theabove section 1-3-1 (Table 3). As a result, it was found that thepercentage of hIgG+_hVEGF-A− was increased in comparison to the negativecontrol (CS(−)) in the presence of Wortmannin, A66, PI3Kα Inhibitor 2,PF-4989216, GSK1059615, PI-103, and PIK-90. It became clear that, notonly in a cell line expressing an anti-Sema3A antibody (cIgM), but alsoin a cell line expressing an antibody (hIgG) reacting against VEGF-Athat was an antigen different from Sema3A, sequence changes werepromoted by the PI3Kα inhibitors.

1-3-6. Diversification-Promoting Effects of Pi3Kα Inhibitors in AffinityMaturation Using Human Antibody-Generating Cell Lines

Subsequently, using A66 and PI3Kα Inhibitor 2 having the highestdiversification-promoting effects in the above section 1-3-5, cultureconditions for promoting Affinity Maturation were studied to both in thehVEGF-A−#33 cell line and in the hVEGF-A−#44 cell line. The hVEGF-A−#44cell line is a DT40 cell clone expressing an anti-hVEGF-A antibody(hIgG), as with the hVEGF-A−#33 cell line. As described in the abovesection 1-2-6, the cells were cultured in the presence or absence of aPI3Kα inhibitor (A66: 10 μM, PI3Kα Inhibitor 2: 500 nM). After the cellshad been cultured under individual culture conditions, the culturedcells were stained with AF647-labeled hVEGF-A and Goat Anti-HumanIgG-PE, and were then subjected to FCM analysis. In the hIgG+_hVEGF-A+cell population, a gate (Affinity Maturation Gate) was established, sothat cells with improved binding ability to the antigen hVEGF-A (5 ormore times increased in MFI) could be comprised (FIG. 9 ), and acomparison was then made in terms of the percentage of the cells in thegate. As a result, it was found that the percentage of the cells withimproved antigen-binding ability was increased under conditionscomprising A66 and PI3Kα Inhibitor 2, rather than under conditions notcomprising such drugs (FIG. 10 ). These results suggest that the numberof clones with improved antigen-binding ability was increased as aresult that the PI3Kα inhibitors promoted sequence changes in theantibody variable regions of human IgG reacting against hVEGF-A. That isto say, it became clear that sequence changes in the antibody variableregions were promoted by performing Affinity Maturation under conditionscomprising A66 and PI3Kα Inhibitor 2, and thus that positive clones withimproved antigen-binding ability could be efficiently obtained.

1-3-7. Analysis of Antibody Variable Region Gene Sequences of Cloneswith Improved Antigen-Binding Ability According to Affinity Maturation,and Measurement of Antibody Affinity

Furthermore, from the hVEGF-A−#33 cell line and the hVEGF-A−#44 cellline that were cultured under individual culture conditions described inthe above section 1-2-6, the top 0.2% cells of a cell population withimproved antigen-binding ability were isolated according to FACS. Theantibody variable region gene sequences of individual growing cloneswere decoded, and the obtained nucleic acid sequences were thenanalyzed. As a result, it was found that a wide variety of sequencechanges occurred in 95% or more of the obtained clones and also, changesin the amino acid sequences associated therewith occurred. Moreover, itwas also confirmed that, among such clones, there were clones withimproved affinity (KD value) in comparison to the original cloneantibody.

From these results, it was confirmed that sequence changes in antibodyvariable region genes occurred in clones with improved antigen-bindingability, and accordingly, changes in the amino acid sequences and theimprovement of affinity also occurred.

Industrial Applicability

The present invention relates to a method for promoting diversificationof the amino acid sequences of variable regions of an antibody generatedby an avian B cell population. Therefore, it is expected that thepresent invention will be utilized in the field of medicine and pharmacythat are associated with antibody drugs, or in the field of usingantibodies as research reagents.

1. (canceled)
 2. A method for promoting diversification of amino acidsequences of variable regions of an antibody generated by an avian Bcell population, wherein the method comprises: contacting a populationof avian B cells with a PI3Kα inhibitor to diversify amino acidsequences of variable regions of an antibody generated by said avian Bcell population; and sequencing amino acid sequences of variable regionsof the antibodies generated by said avian B cell population after saidcontact with said PI3Kα inhibitor to confirm diversification of aminoacid sequences of variable regions of said antibodies generated by saidavian B cell population after contact with said PI3Kα inhibitor.
 3. Amethod for producing an antibody comprising performing the method ofclaim 2 and isolating an antibody having a diversification of amino acidsequences of variable regions after said sequencing.
 4. An antibodycomplementarity determining region (CDR) sequence of an antibody,wherein the CDR sequence is obtained by the method according to claim 2.5. An antibody variable region sequence obtained by the method accordingto claim 2.